Confocal microscopy with multi-spectral encoding

ABSTRACT

A scanning confocal microscopy system, especially useful for endoscopy with a flexible probe which is connected to the end of an optical fiber( 9 ). The probe has a grating( 12 ) and a lens( 14 ) which delivers a beam of multi-spectral light having spectral components which extend in one dimension across a region of an object and which is moved to scan in another dimension. The reflected confocal spectrum is measured to provide an image of the region.

This application is a 371 of PCT/US99/04356 filed Feb. 26, 1999 whichclaims the priority benefit of U.S. Provisional Application No.60/076,041, filed Feb. 26, 1998.

DESCRIPTION

The present invention relates to systems (method and apparatus) forconfocal microscopy for the examination or imaging of sections of aspecimen of biological tissue, and particularly to such systems usingmulti-spectral illumination and processing of multi-spectral light.

Currently, the use of fast scanning confocal microscopy is limited toaccessible surfaces of the skin and the eye. The reason for this is thatthe only reliable methods for optical scanning must be performed in freespace. In addition, the size of these optical scanners prohibit theiruse in small probes such as endoscopes or catheters. It is a feature ofthe invention to miniaturize the fast scanning mechanism and increasethe number of medical applications of confocal microscopy to include allsurfaces of the body, gynecologic applications, probe-basedapplications, and internal organ systems.

Multi-spectral light was proposed for use in confocal microscopy, butonly for imaging vertically-spaced regions of a body under examination.See B. Picard, U.S. Pat. No. 4,965,441, issued Oct. 25, 1990. Aninterferometer using a grating to obtain multi-spectral light which isresolved in the interferometer to obtain a spectroscopic image isdisclosed in A. Knuttal, U.S. Pat. No. 5,565,986, issued Oct. 15, 1996.A lens having a color separation grating which obtains a multi-spectrallight is disclosed in U.S. Pat. No. 5,600,486, issued Feb. 4, 1997. Suchmulti-spectral proposals are not effective for high resolution imagingusing a compact, flexible probe. A confocal microscope system accordingto this invention can be miniaturized and incorporated into a compactprobe. In addition, by allowing light delivery through a single opticalfiber, the probe may also be easily incorporated into catheters orendoscopes. Thus, a confocal microscope in accordance with the inventionallows imaging of all accessible surfaces of the body and increases thebiomedical applications of confocal microscopy by an order of magnitude.

Briefly described, a confocal microscopy system embodying the inventionilluminates a region of interest in a body into which said probe may beinserted with a confocal spectrum extending along one dimension. Opticsin said probe or physical movement of said probe enabled by attachmentthereto of a flexible light conductive member (which may be an opticalfiber), enables scanning of said spectrum along one or two additionaldimensions thereby providing for two or three dimensional imaging of theregion. The reflected confocal spectrum may be detected or decodedspectroscopically, preferably with a heterodyne detection mechanismwhich may be implemented interferometrically.

The invention will be more apparent from the following drawings wherein

FIG. 1 is a schematic diagram of a spectrally encoded confocal probe inaccordance with the invention where specific wavelengths are shown forillustrative purposes, their exact values depending on the opticalparameters of the system.

FIG. 2 is a plot of spectrally encoded light obtained by confocaldetection using direct spectral detection in accordance with thisinvention, where different wavelengths are detected by turning thespectrometer grating.

FIG. 3 is a schematic diagram showing a system embodying the inventionusing a spectrometer for measurement of the spectrum, I(λ), whichcorresponds to reflectance from different transverse locations, x, onthe specimen.

FIG. 4 is a schematic diagram of a system embodying the invention havingspectrally encoded confocal detection using interference spectroscopy.

FIG. 5A-D are schematic diagrams showing: (a) image formation; (b)translation of the optical fiber in the y direction; (c) rotation of theoptical fiber in the forward firing mode; and (d) rotation of theoptical fiber in the side firing mode.

FIG. 6 is a schematic diagram showing cross-sectional image formation byscanning the optical fiber or the objective lens along the z axis usinga system embodying the invention.

FIG. 7 is another schematic diagram of a system embodying the inventionwherein optical zoom is achieved by moving the focus of an intermediatelens in and out of the image plan of the objective.

Referring now to the figures, multi-spectral encoding for confocalmicroscopy uses a broad bandwidth source 10 as the input to themicroscope. In the probe 8 of the microscope, the source spectrumprovided via an optical fiber 9 is dispersed by a grating 12 and focusedby an objective lens 14 onto the sample 16. A lens 9 a is preferablydisposed between the optical fiber 9 and the grating 12 to collimate thelight from the optical fiber, as shown in FIG. 1, however, lens 9 a maybe removed. The spot for each wavelength is focused at a separateposition, x, on the sample (FIG. 1). The reflectance as a function oftransverse location is determined by measuring the reflected confocalspectrum from the sample 16 returned from probe 8.

The number of wavelengths or points that may be resolved is determinedby: $\begin{matrix}{{\frac{\lambda}{\delta \quad \lambda} = {mN}},} & (1)\end{matrix}$

where λ is the center wavelength, δλ is the bandwidth of the spectrum, Nis the number of lines in the grating 12 illuminated by thepolychromatic input beam 10, and m is the diffraction order. If thetotal bandwidth of the source is Δλ, the number of resolvable points, nis defined by: $\begin{matrix}{n = \frac{\Delta \quad \lambda}{\delta \quad \lambda}} & (2)\end{matrix}$

For an input source with a center wavelength of 800 nm, a bandwidth of25 nm, an input spot diameter of 5 mm, a diffraction grating of 1800lines/mm and a diffraction order of 1, n=281 points may be resolved bythe spectrally encoded confocal system (FIG. 2). The parameters used inthis example may be found in common, inexpensive optical components. Thenumber of points may be increased by simply increasing the input spotdiameter or the bandwidth of the source. Increasing the spot diameterincreases the resultant probe diameter. Increasing the bandwidth of thesource could be accomplished by using a broader bandwidthsuperluminescent diode, a rare earth doped fiber superfluorescentsource, or a solid state modelocked laser.

Consider next the multi-spectral process. First, consider directspectral measurement. The reflectance from the sample 16 as a functionof transverse location is determined by measuring the reflected confocalspectrum from the sample arm 18. The spectrum may be measuredefficiently by incorporating the probe 8 in the sample arm of aMichelson interferometer 20 (FIG. 3) and detecting the light transmittedthrough a high resolution spectrometer 21 at the output port 19 of theinterferometer. Thus, each wavelength measured corresponds to a separateposition, x, on the sample (FIG. 3). The advantage to this method overtraditional real time confocal microscopy is that the fast axis scanning(˜15 kHz) may be performed external to the probe 8 by the spectrometer21 with approximately 0.1 nm spectral resolution for the parametersgiven above, well within reach of high quality spectrometers.

High sensitivity may be achieved through the use of the heterodynedetection. If the reference arm 22 is modulated, such as by modulator 23with mirror 24 (FIG. 3), the interference of light from the sample arm18 and the reference arm 22 will also be modulated. High signal-to-noiseratios may be then achieved by lock-in detection on the reference armmodulation frequency of detector 26.

Another method for measuring the spectrum is interference or Fouriertransform spectroscopy. This may be accomplished by inserting a linearlytranslating mirror 28 in the reference arm 22 and measuring thecross-correlation output 30 from the interference spectrometer due tothe interference of the reflected light from the sample and referencearms 18 and 22, respectively (FIG. 4). The advantages to this type ofspectroscopic detection include the ability to achieve higher spectralresolutions than direct detection methods, efficient use of the returnedlight, inherent modulation of the reference arm 22 by the Doppler shiftof the moving mirror 28, and the capability to extract both reflectanceand phase data from the sample 16. The ability to extract phase datafrom the sample may allow detection of refractive index as a function oftransverse position, x, which is useful to reveal the molecularcomposition of the sample as well as provide an additional source ofimage contrast other than the reflectivity of the sample specimen 16.Finally, interferometric detection has the potential to allowelimination of high order multiple scattering from the confocal signalby coherence gating.

Consider finally image formation. The multi-spectral encoding of thetransverse location, x, allows the performance of a one-dimensionalraster scan. To obtain an image, a scan of another axis must beperformed, which is usually slower. Methods of accomplishing this slowscanning of the y axis include moving the optical fiber 9 in the ydirection (FIG. 5B), or rotating the entire probe 8 around the opticalfiber axis either in a forward scanning configuration (FIG. 5C) or aside-firing configuration (FIG. 5D). Cross-sectional images may becreated by scanning the optical fiber 9 or the objective lens 14 alongthe z axis (FIG. 6). Finally, a zoom mode may be created by scanning theoptical fiber 9 (or another lens 32 between grating 12 and objectivelens 14), in and out of the image plane of the objective lens (FIG. 7).Both linear motion along the y or z axis and rotation are easilyaccomplished in a compact probe by use of piezoelectric transducers. Asshown in FIG. 5A, signals may be received by a computer 34 fromspectroscopic detector 32 by a spectrometer (such as described inconnection with FIG. 3) or Fourier transform (such as describedconnection with FIG. 4) representing an image of the a microscopicsection of the sample, and the image displayed on a display coupled tothe computer.

From the foregoing description, it will be apparent that the inventionprovides a confocal microscopy system which (a) is compact, opticalfiber-based, capable of enabling confocal microscopy through a flexiblecatheter or endoscope; (b) is fast-scanning which takes place externalto the probe; (c) allows phase information to be retrieved; and (d)provides a number of resolvable points proportional to the bandwidth ofthe source and the beam diameter on the grating. Variations andmodifications in the herein described confocal microscopy system inaccordance with the invention will undoubtedly suggest themselves tothose skilled in the art. Accordingly, the foregoing description shouldbe taken as illustrative and not in a limiting sense.

What is claimed is:
 1. A confocal microscope system which comprises aprobe movable into a body region of interest, said probe having meansfor illuminating said region with a confocal spectrum of light extendingalong one substantially transverse dimension, means for obtaining animage of the region of the specimen by moving said spectrum alonganother dimension and measuring the reflected confocal spectrum oflight.
 2. The system according to claim 1 wherein said probe is mountedon the end of a flexible, light-conducting member.
 3. The systemaccording to claim 2 wherein said member is an optical fiber.
 4. Thesystem according to claim 3 wherein said fiber is rotatable ortranslatable to move said probe in said another dimension.
 5. The systemaccording to claim 1 wherein said means for moving said spectrumcomprises means for moving an image plane containing said spectrumoptically or by physically moving said probe.
 6. The system according toclaim 5 wherein said probe is moved physically to scan said spectrum insaid another dimension and said probe has means for optically movingsaid image plane to scan in still another direction, thereby enabling3-D imaging.
 7. The system according to claim 1 wherein said means forobtaining said image comprises heterodyne detection means.
 8. The systemaccording to claim 7 wherein said heterodyne detection means includes aninterferometer.
 9. The system according to claim 8 wherein saidinterferometer has a sample arm terminated by said probe, a referencearm terminated by a mirror, an output arm having a spectroscopicdetector, an input arm having a source of polychromatic illumination,and a beam splitter for directing light from said source to said sampleand reference arms and directing interfering light containing saidreflected confocal spectrum into said output arm.
 10. The systemaccording to claim 9 wherein said reference arm includes means formodulating said reflected spectrum.
 11. The system according to claim 10wherein said modulating means comprising means for reciprocallyoscillating said mirror or a modulator.
 12. The system according toclaim 11 wherein said modulator or reciprocal oscillation is at acertain frequency, and means for lock-in operation of said detector atsaid frequency.
 13. The system according to claim 9 wherein saiddetector is a spectrometer.
 14. The system according to claim 9 whereinsaid detector includes a cross-correlator or a Fourier transformspectrometer.
 15. The system according to claim 1 wherein said probecomprises a grating and an objective which provides said confocalspectrum in an image plane of said objective.
 16. The system accordingto claim 15 wherein said probe is sufficiently small size to beinsertable into an organ internal of said body.
 17. A system forconfocally imaging tissue comprising: a source for producing light;means for producing a confocal spectrum of said light; means forfocusing said confocal spectrum in a direction into said tissue defininga first dimension and receiving returned light from said tissue, inwhich said confocal spectrum producing means is capable of providing aconfocal spectrum which when focused by said focusing means extendsalong a second dimension in said tissue different from the firstdimension; and means for detecting said returned light in accordancewith a spectrum of said returned light to provide an image representingsaid tissue.
 18. The system according to claim 17 further comprisingmeans for scanning said confocal spectrum in at least one dimension withrespect to said tissue.
 19. The system according to claim 17 wherein atleast said producing means and said focusing and receiving means arelocated is a probe capable of insertion in a body.
 20. The systemaccording to claim 17 further comprising an optical fiber which providessaid light from said source to said producing means, and provides saidreturned light from said focusing and receiving means to said detectingmeans.
 21. The system according to claim 17 wherein said producing meansand focusing means are provided by more than one optical element. 22.The system according to claim 17 wherein said detecting means comprisesat least a spectrometer.
 23. The system according to claim 22 furthercomprising interferometric means for enabling said detecting means. 24.The system according to claim 17 wherein said light is polychromatic,said focusing means provides for focusing said confocal spectrum intosaid tissue along multiple positions in the tissue encoded in accordancewith characteristics of the polychromatic light and said confocalspectrum producing means, and said detecting means spectroscopicallydetects said returned light to provide an image of a section of thetissue in accordance with the encoded positions of the confocal spectrumfocused in the tissue.
 25. The system according to claim 17 wherein saidsecond dimension is substantially transverse with respect to said firstdimension.
 26. A method for confocally imaging tissue comprising thesteps of: providing a source of polychromatic light; producing aconfocal spectrum of said light with the aid of a diffractive element;focusing said confocal spectrum into said tissue along multiplesubstantially transverse positions in the tissue encoded in accordancewith characteristics of the polychromatic light and said diffractiveelement; receiving returned light from the tissue; and spectroscopicallydetecting said returned light and producing an image of a section of thetissue in accordance with the encoded positions of the confocal spectrumfocused in the tissue.
 27. A system for imaging tissue comprising: adiffractive element capable of providing illumination of one or morewavelengths along a first dimension; and a lens which focuses saidillumination in a direction into said tissue along a second dimensiondifferent from said first dimension, and said lens receives returnedillumination from said tissue representative of one or more locations insaid tissue in accordance with said one or more wavelengths.
 28. Thesystem according to claim 27 further comprising a probe comprising atleast said lens and said diffractive element.
 29. The system accordingto claim 27 wherein said first dimension is substantially transversewith respect to said second dimension.
 30. The system according to claim27 wherein said lens focuses said illumination into one or more spots inthe tissue at said one or more locations in accordance with said one ormore wavelengths.
 31. The system according to claim 27 furthercomprising means for scanning said tissue with said illumination focusedby said lens.
 32. The system according to claim 31 further comprisingmeans for detecting said returned light to provide an image of saidtissue representative of said region of said tissue.